The most important findings of the study are that CM derived from hUC-MSC reduced OA progression-related matrix degradative-related factors and increased OA prevention-related matrix factors. Furthermore, it was confirmed that hUC-MSC CM inhibited the expression of chondrocyte-derived E-selectin, thereby reducing the possibility of contact between monocytes and chondrocyte-derived E-selectin.
According to their structure and substrates, MMPs can be categorized into several types, including collagenase (MMP-1, MMP-13), gelatinase (MMP-2, MMP-9), stromelysin (MMP-3), metalloelastase (MMP-12), matrilysin (MMP-7), and membrane- type matrix metalloproteinase (MT-MMPs) (33). The results of this study indicate that the expression of MMP-1, MMP-3, and MMP-13 was significantly reduced by hUC-MSC CM. Considering the fact that collagen is the most abundant structural macromolecule in cartilage and that it makes up approximately 60% of cartilage's dry weight(42), the fact that MMP-1 and MMP-13 belonging to the collagenase category were significantly reduced by hUC-MSC CM is undoubtedly a remarkable result. In particular, the result that MMP-13, which degrades Type Ⅱ collagen, which comprises 90 to 95% of all collagen (42), decreased suggests that hUC-MSC CM
４３
could be an excellent therapeutic agent for alleviating OA progression. Furthermore, considering that one of the causes of bone destruction in osteoarthritis is an imbalance of MMPs and their inhibitor, TIMP, the result of significant increase in TIMP-1 and TIMP-3 by hUC-MSC CM suggests that it could be used as a therapeutic agent in dealing with the biological cause of the disease.
Therefore, it may be concluded that hUC-MSC CM exhibits anti- inflammatory effects by reducing the expression of factors associated with cartilage degradation.
NGS analysis conducted in this study to examine the anti- inflammatory effect of hUC-MSC CM was significantly simplified by using MLDEG to derive and verify more accurate OA-related candidate genes. Prior to machine learning analysis, four independent statistical analysis tools were employed, and since different analysis methods were applied to each tool, passing the filter of four tools alone represents a highly reliable discovery of candidates. Additionally, the MLDEG used in this study has already been validated due to the publication of the study in 2021 (31). As a result, only six genes were filtered through MLDEG, thereby increasing the efficiency of the in vitro validation. In several chondrocyte batches, we verified that the expression of E-selectin was increased by IL-1β and decreased by hUC-MSC CM, the
４４
same results as the NGS analysis, indicating a similar trend has been reported in the Sandell paper published in 2009 (43).
Additional experiments will have to be conducted, however, to identify the quantity of soluble E-selectin released by chondrocytes in supernatants.
In this study, hUC-MSC CMs were obtained from hUC- MSCs that had been treated with IL-1β, creating a similar OA environment where high concentrations of IL-1β are present.
Despite not being reported in the results, higher concentrations of growth factor and anti-inflammatory cytokine were released from IL-1β stimulated hUC-MSCs when compared to hUC-MSCs in the absence of IL-1β stimulation. It is possible to interpret and compare these results with previous studies in other human tissues.
For example, previous studies, such as Broekman (2016), Chen (2015), and Fan (2012), confirmed better therapeutic effects in hMSCs pre-treated with inflammatory substances such as IL-1β or TNF-α. In view of this, it has been proposed that soluble factors released by hUC-MSC following IL-1β stimulation regulate E-selectin expression in chondrocytes exposed to IL-1β.
In our future work, we plan to observe soluble E-selectin in supernatants shed from chondrocytes. In HUVEC cultures, expression of E-selectin is absent at basal levels but is rapidly
４５
(2h) induced by TNF. Surface expression is transient, it declines to 10% of peak levels within 24 hours(44). In light of this, it is predicted that the quantity of E-selectin expressed in chondrocytes will be significantly lower than soluble E-selectin in supernatant. At the same time, the concentration of E-selectin present in shedding and soluble form will be measured as well as the degree of chondrocyte activation, ultimately allowing change in chondrocytes to be noticed. As an additional experiment, the amount of E- selectin present in soluble form will be measured and, at the same time, the degree of monocyte activation and subsequent changes in chondrocytes will be determined.
４６
Chapter 5 Conclusion
hUC-MSC significantly alleviated chondrocyte damage caused by IL-1β -induced inflammation. hUC-MSC CM resulted in a significant reduction in pro-inflammatory cytokines such as TNF-α and COX-2 and matrix degradative enzymes such as MMP-1, MMP-3, MMP-13, and an increase in matrix prevention factors such as TIMP-1, TIMP-3. E-selectin expression in chondrocytes was significantly reduced in the group of hUC-MSC CM, which led to reduced monocyte adhesion. This study demonstrated that hUC-MSC CM exert chondroprotective effects.
The paracrine mechanism of hUC-MSC may be responsible for the protection of chondrocytes against IL-1β exposure. Therefore, the current study offers a potential method for attenuating osteoarthritis by taking a fundamentally biological approach
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국문초록
골관절염 연골세포의 E-selectin 의존성 단핵구 동원 억제를 통한 인간 탯줄유래 중간엽 줄기세포
배양액의 항염증 효과
김 예 솔 의학과 중개의학전공 The Graduate School Seoul National University
배경: 70세 이상 인구의 80% 이상에 영향을 미치는 골관절염은 흔한 질병 중 하나이다. 하지만 현재 통증과 염증 조절을 위한 NSAIDS 약물처방 및 corticosteroid 투여 외에 근본적인 생물학적 원인을 다루는 약물은 현 시 장에 없는 상태이다. 골관절염의 발달과 진행에 대한 면역세포의 관여는 최 근 연구에서 강조되었다. 특히 활성화된 M1 대식세포는 골관절염의 염증과 정을 증폭시키는 사이토카인을 분비한다. 본 연구에서는 NGS분석을 통하 여 IL-1β 환경에 노출된 연골세포의 E-selectin 발현이 증가됨을 확인하 였으며, 이는 라이간드를 발현하는 주변 단핵구를 IL-1β 병변 환경으로 동원한다. 단핵구는 골관절염 환자의 체내 존재하는 IL-1β 및 여러 염증 성 사이토카인들에 의하여 염증성 물질을 방출하는 M1 대식세포로 분극화 될 수 있다. 본 연구는 면역억제 세포로서 항염증 인자 방출을 통해 염증을 감소시키고 단핵구의 활성도를 감소시킬 수 있는 인간 탯줄(human
５１
Umbilical Cord, hUC) 유래 중간엽 줄기세포(Mesenchymal Stem Cell, MSC)를 배양한 배양액(Conditioned Medium, CM)이 골관절염 염증상황 에서 연골세포의 E-selectin의 발현을 감소시켜 E-selectin 의존성 단핵 구 모집을 억제한다는 가설과 함께 골관절염 진행 억제능력을 평가하였다.
방법: 슬관절 치환술을 시행한 총 7명의 만성 골관절염 환자에서 연골세포 를 분리하였으며 hUC-MSC 분리 위하여 제왕절개 시술을 받는 산모에게 서 탯줄을 분리, 수령하였다. 분리한 hUC-MSC에 IL-1β 자극을 준 후 얻 어낸 CM을 IL-1β 환경에 노출 되어있는 연골세포에 처리해 주었다. 최종 실험그룹은 컨트롤, IL-1β, CM 그룹으로 진행되었으며 IL-1β병변 환경 에 미치는 CM의 영향을 확인하기 위하여 연골세포의 유전자와 단백질 수 준에서 염증성 및 항염증성 사이토카인, 기질 효소 및 이들 효소의 억제제 의 발현을 조사하였다. 후에 MLDEG 분석 방법을 통하여 IL-1β 환경에 노 출된 연골세포와 IL-1β 환경에 노출된 후 hUC-MSC CM을 처리해준 연 골세포 비교 시 발현 정도에 차이를 나타내는 유전자를 비교 분석하였다.
결과, 전염증성 후보 유전자 5개, 항염증성 후보 유전자 1개를 얻었다. 최종 적으로 단핵구 모집에 관여하는 SELE 유전자를 선택, IL-1β 환경에 노출 된 연골세포와 IL-1β 환경에 노출된 후 hUC-MSC CM에 추가 노출된 연 골세포 에서의 E-selectin 발현 정도를 비교하였다. 또한 컨트롤, IL-1β, CM 3개 그룹의 연골세포와 단핵구를 1시간 direct co-culture하여 연골 세포 발현 E-selectin 의존성 단핵구의 부착 정도를 확인하여 hUC-MSC CM의 항염증 효과를 평가하였다.
결과: IL-1β 환경에 노출된 연골세포에 hUC-MSC CM 처리시 기질분해
５２
효소인 MMP1, MMP3, MMP13, ADAMTS4 단백질 발현이 차례로 47.4%, 31.6%, 21.8%, 그리고 44.9% 감소, 기질 분해 방지 인자인 TIMP1, TIMP3 단백질 발현이 각각 128.9%, 그리고 68.4% 증가함을 확 인하였다. MLDEG 분석 방법을 동원하여 IL-1β 와 CM 그룹의 연골세포 에서 발현 차이를 보이는 총 126개의 전염증성 유전자와 238개의 항염증 성 유전자를 발견하였다. 후에 combined p-value <0.02, combined logFC>1 그리고 기존의 사전 지식을 필터링 조건으로 설정하여 추가 분석 한 결과 전염증성 유전자 5개(CXCL10, SELE, CAGE1, MYB, and IP6K3), 항염증성 유전자 1개(PTPRF)를 최종적으로 얻었다. 단핵구 모 집에 관련성이 있어 OA 진행에 영향을 미치는 SELE 유전자를 추가 실험 을 위한 최종 유전자로 선발하였다. 각 환경에 따른 연골세포에서의 E- selectin 발현양을 측정한 결과 hUC-MSC CM에 의한 E-selectin발현이 IL-1β그룹의 연골세포 대비 단백질 수준에서 21.8% 감소됨을 확인하였다.
각 환경의 연골세포에서 발현되는 E-selectin양에 따른 단핵구와의 결합 정도를 확인한 결과, IL-1β그룹의 연골세포 대비 CM그룹에서 단핵구와의 결합이 평균 53.9% 감소하였다. 더 나아가 연골세포의 E-selectin 발현을 blockade사용하여 차단 후 단핵구와 결합시킨 결과 blockade 사용하지 않 은 그룹 대비 IL-1β그룹에서 42.1%, CM그룹에서 41.8% 감소함을 확인 하여 단핵구가 E-selectin과 결합함을 확인하였다.
요약: OA 발병으로 인하여 고농도의 IL-1β환경에 노출된 연골세포에서의 E-selectin 발현은 염증성 물질 방출가능한 M1 대식세포로 분극화가 가 능한 주변 단핵구를 병변환경으로 동원하게 된다. hUC-MSC CM은 연골